Certain embodiments relate generally to the field of medicine. More particularly, certain embodiments concern compositions and methods for ameliorating chemotherapy-induced infertility.
Spermatogonial stem cells (SSCs) are adult stem cells found in the mammalian testis that are responsible for maintaining spermatogenesis throughout adult life. Spermatogenesis is a highly productive process, producing millions of sperm each day (1000 sperm/heart beat). Because of this high degree of proliferation, spermatogenesis is susceptible to chemotherapy treatments, which can lead to long-term or permanent infertility or reduced fertility. SSCs themselves do not divide frequently, but high doses of various chemotherapeutic agents have been shown to kill SSCs, the loss of which would result in permanent loss of spermatogenesis and male infertility.
Adult males who will undergo potentially-sterilizing chemotherapy can prospectively cryopreserve sperm from a semen sample for later use in the fertility clinic via in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). However, prepubertal boys who are not yet producing mature gametes cannot take advantage of this standard-of-care option for preserving their future fertility. This is a significant problem because the overall event-free survival rate for childhood cancers is approaching 80%, which enables patients to look beyond cancer to a productive life after cure. Moreover, parenthood is important to cancer survivors and distress over infertility can have long-term psychological and relationship implications.
To address this clinical need, several experimental technologies are on the horizon that may facilitate preserving the future fertility of prepubertal boys. Spermatogonial stem cell (SSC) transplantation is one experimental approach that may have application for preserving and restoring fertility of prepubertal boys. With this technology, SSCs would be harvested from the patient surgically prior to chemotherapy, cryopreserved, and stored until their reintroduction into the patient's testis sometime after chemotherapy treatment. This approach has the potential to regenerate spermatogenesis in these patients who currently have no other option to preserve their future fertility. Feasibility of this approach is supported by results in animal models including rodents, pigs, goats, bulls, dogs, and monkeys.
However, transplantation of cryopreserved testis cells isolated from patients with cancer carries an inherent risk of reintroducing contaminating malignant cells back into patients. Using rodent models, there are conflicting reports about the feasibility of separating SSCs from malignant cells using immune-based approaches and the current data for eliminating cancer cells from contaminated primate and human testicular cell suspensions is contradictory. Moreover, another major limiting factor to successful application of SSC transplantation in the clinic is that the number of SSCs which can be prospectively isolated from patients before treatment is lower than the number needed for successful SSC transplantation at a later date. Thus, while spermatogonial stem cell transplantation has proven effective for regenerating spermatogenesis and fertility in small and large animal models, clinical translation will likely lag until methods are developed to eliminate the risk of re-introducing malignant cells into a cancer survivor and isolate or amplify sufficient SSCs for transplant.
Testicular tissue xenografting is an alternative technique that may provide a therapeutic option for prepubertal cancer patients and avoids the risk of malignant cell contamination. Using this approach, intact testicular tissue grafts from immature mice, rats, hamsters, pigs, goats, and nonhuman primates were competent to produce complete spermatogenesis following ectopic transplantation under the skin of mouse hosts. Sperm retrieved from rodent grafts (freshly transplanted or cryopreserved) could be used for intracytoplasmic sperm injection (ICSI) to produce offspring. However, to date, there has only been one report of sperm production in grafts of cryopreserved prepubertal rhesus macaque testicular tissue from among numerous studies using monkey and primate tissue. Moreover, little is known about the risk of zoonotic disease transmission from germ cells derived from tissue transplanted in a xeno intermediate. Alternatively, grafts could be implanted back into the patients in the homotopic site (within the testis) or at a heterotopic site such as beneath the skin, but this approach bears the same risk of malignant cell contamination as SSC transplantation. Alternate strategies involving gamete production in vitro from cultured cells or tissue may provide options for fertility restoration for some patients, but their utility/efficacy have yet to be proven.
Yet another alternative approach would be to prospectively preserve SSCs from the toxicity of chemotherapy in their native testicular environment. Along those lines, several studies have investigated the use of hormone treatments to suppress the gonadotropins (i.e., FSH and LH). This approach reduces intratesticular testosterone levels, which protects testicular somatic cells and enhances the recovery of spermatogenesis from surviving SSCs. Prompted by these promising observations in lab animals, seven clinical trials tested this approach in adult humans, all but one study failed to demonstrate an improvement in sperm counts after gonadotropin suppression. Subsequently, gonadotropin suppression has received little attention as an option for male fertility preservation.
One previous study in mice from Kim and colleagues (Andrologia (2010) 43:87-93) reported similar protection of spermatogenesis in irradiated mice using granulocyte colony stimulating factor (G-CSF). In that study, mice were treated with G-CSF (100 μg/kg/day) for 3 days prior to 5 Gy of testicular gamma irradiation and the effects on spermatogenesis were measured 3 weeks later. The results demonstrate G-CSF treatment induced a modest improvement in the numbers of surviving differentiated spermatogonia following sub-sterilizing irradiation. While radiation and alkylating chemotherapy treatment are both used to kill malignant cells because they both target rapidly dividing cells, they have different mechanisms of action and their effects on spermatogenesis are different. By extension, methods used to protect spermatogenesis do not always have the same beneficial effects for radiation and chemotherapy insults. Indeed, other previous studies examining the use of gonadotropin suppression to protect spermatogenesis from cytotoxic insult demonstrated that beneficial effects in irradiated animals do not translate to chemotherapy. Specifically, rats treated with GnRH antagonists prior to sterilizing irradiation showed improved spermatogenic regeneration, while similar treatments prior to busulfan chemotherapy failed to promote spermatogenic regeneration. Thus, the prior results with irradiation cannot be extrapolated to nor are they predicative of G-CSF amelioration of chemotherapy-induced infertility.
Of note, the radiation dose employed in the prior art (5Gy irradiation+G-CSF) is considered sub-sterilizing, and thus, spontaneous cell survival is expected, particularly among the differentiated spermatogonia. Moreover, the time to analysis after G-CSF treatment and irradiation (21 days) measures effects at the level of differentiated Type-B spermatogonia, not stem cells. Specifically, the duration of spermatogenesis in mouse is 40.5 days (the amount of time required for a differentiating division of an a single spermatogonial stem cell to produce spermatozoa in the testis, and thus, assessment of stem cell survival or repopulation requires a longer time-frame for analysis. Indeed, bona fide assessment of spermatogenic regeneration from stem cells requires observation of complete spermatogenesis, which can only be appropriately evaluated at ˜2 months following treatment to give sufficient time to allow exit of damaged spermatogonia from the testis and allow progeny from stem cells to differentiate beyond the latest stages of mitotic spermatogonia (i.e., prior to meiotic entry and spermiogenesis in haploid spermatids).